Sphingosine-1-phosphate (S1P) is a potent sphingolipid mediator that regulates diverse cellular processes important for cancer progression including cell growth and survival, invasion, angiogenesis, lymphocyte trafficking, and inflammation, among others. Although S1P is produced inside cells by two closely related sphingosine kinases, SphK1 and SphK2, most of the research to date in the S1P field has been concentrated on its actions as a ligand for the five S1P receptors, and prior to our work, no intracellular targets had been recognized. We recently identified HDAC1/2 as the first direct intracellular targets of S1P, and demonstrated that S1P, produced by nuclear SphK2, is an endogenous small molecule inhibitor of these enzymes, linking nuclear S1P to epigenetic regulation of gene expression. In preliminary studies, we have discovered that TNF receptor-associated factor 2 (TRAF2), a key component in activation of NF-kB, is another novel intracellular target of S1P. Our data indicates that S1P may be the missing cofactor for the E3 ubiquitin ligase activity of TRAF2, suggesting a new paradigm for regulation of NF-kB, a transcription factor important for cancer and inflammation. In this proposal, we will examine the hypothesis that the location of S1P production dictates its functions and that S1P is not only a ligand for the S1PRs but also serves as an intracellular molecular switch that regulates the activity of its targets, HDAC1/2 and TRAF2, important for cancer and inflammation. In the first aim, we will determine the role of S1P produced by SphK2 in the nucleus as an endogenous regulator of HDACs. In the second, we will determine the role of S1P produced by SphK1 as a direct regulator of the E3 ligase activity of TRAFs, lysine 63-linked polyubiquitination, and NF-kB signaling. The third aim will address the importance of S1P and its regulation of HDAC1/2 and TRAF2 in a mouse model of chronic inflammation leading to cancer. These three aims represent a novel and unique approach to understanding S1P biology and its multi-faceted actions and will open new areas of research. This proposal provides a paradigm shift from inside-out signaling by S1P to include important signaling inside cells and will provide evidence that S1P is one of the critical factors that bridges chronic inflammation to tumor promotion and progression. PUBLIC HEALTH RELEVANCE: The bioactive sphingolipid metabolite S1P and the kinases that produce it, SphK1 and SphK2, have emerged as critical regulators of numerous fundamental biological processes important for health and disease, particularly cancer and inflammation. It is difficult to find an area of cell biology and physiology in which S1P does not have important if not key roles. This proposal will be a major contribution towards understanding the enigmatic nature of the pleiotropic actions of S1P, modifying the focus of research on S1P signaling at the cell surface to include signaling inside cells, and will have important clinical applications for inflammation and cancer.